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Abstract:
Site-directed fluorescence labeling and voltage-clamp fluorometry has been used in our laboratory to study conformational dynamics of the Na+ /K+-ATPase, leading to the identification of enzyme regions (including the M5-M6 loop and the β-subunit), which participate in the major E1(P)–E2(P) conformational change (Geibel et al. 2003. PNAS. 100:964–969; Dempski et al. 2005. J. Gen. Physiol. In press). Therefore, Na+/K+-ATPase constructs carrying single extracellular cysteines are expressed in Xenopus oocytes and sulfhydryl-specific labeling is performed with tetramethylrhodamine-6-maleimide. Voltage steps applied under Na+/Na+ exchange conditions evoke characteristic fluorescence changes, which are directly correlated to the voltage dependence of transient charge movement that occurs in conjunction with E1P–E2P conformational changes. Here we utilize the Na+ /K+-ATPase N790C mutant and the L311C mutant (which serves as a sensor for the involvement of the extracellular M3-M4 loop in the E1P–E2P reaction). To investigate the influence of [Na+]i, we coexpressed these constructs with the epithelial Na+ channel ENaC to effectively control [Na+]i. When mutant N790C was studied under Na+/Na+ exchange conditions, the time courses of the fluorescence responses for a given voltage jump showed a monoexponential behavior at any [Na+]i. A saturating dependence of amplitudes on [Na+]i with a half-maximal concentration of 7.0 (±2.1) mM was found, and also the kinetics at depolarizing potentials were affected. In contrast, fluorescence responses to voltage steps for mutant L311C were biexponential. A slow phase dominated at low [Na+]i but successively decreased in favor of a fast phase (half-maximal concentration 8.8 [±0.6] mM), which at saturating [Na+]i followed the well-known kinetic behavior of transient charge movements that occur during extracellular Na+ release/rebinding in conjunction with the E1P–E2P conformational change. These results demonstrate that voltage-clamp fluorimetric measurements can be used to study kinetic effects of (weakly or nonelectrogenic) [Na+]i-dependent steps on electrogenic partial reactions of the Na+ pump.
